On the heritability of spontaneous otoacoustic emissions: A twins study. Dennis McFadden *, John C. Loehlin

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1 ELSEVIER Hearing Research 85 (1995) HBIRIrlC, RESEARCH On the heritability of spontaneous otoacoustic emissions: A twins study Dennis McFadden *, John C. Loehlin Department of Psychology and The Institute for Neuroscience, Mezes Hall 330, The Unit,ersity of Texas, Austin, TX USA Received 7 April 1994; revised 13 February 1995; accepted 21 February 1995 Abstract Spontaneous otoacoustic emissions (SOAEs) were measured in human monozygotic (MZ) and dizygotic (DZ) twins and in a sample of non-twins. The number of SOAEs exhibited was more highly correlated in MZ co-twins than in same-sex DZ co-twins. Model-fitting to the correlations suggested that about three-quarters of the individual variation in the expression of SOAEs is attributable to genes. There was no convincing evidence for the heritability of specific SOAE frequencies. In accord with past surveys, SOAEs were more numerous in right than left ears, and in female than male subjects. Also investigated were the numbers of SOAEs exhibited by dark- versus light-eyed people and by MZ versus DZ twins. Those differences in our data were small and not statistically significant, but they were in a direction consistent with other studies: more SOAEs in dark-eyed individuals and in MZ twins. The view presented here is that SOAEs themselves are unlikely objects for natural selection, and probably are epiphenomena resulting from selection for those cochlear mechanisms that contribute to good hearing sensitivitywhich is related to SOAE expression. It is argued that, in addition to genetics, other factors have the potential to affect the specific numbers of SOAEs that are expressed. For example, some aspects of the complex prenatal process of producing a male fetus are presumed to be responsible for the smaller number of SOAEs seen in males than females. Keywords: Spontaneous otoacoustic emissions; Heritability; Prenatal auditory effects; Hearing sensitivity; Sex differences; Ear differences; Twins 1. Introduction Normal cochleas have the ability to produce sounds as well as analyze them. These otoacoustic emissions (OAEs) propagate back through the middle-ear system and escape into the outer ear canal where they can be detected using specialized microphone systems (Kemp, 1978, 1979; Zurek, 1981; see Probst et al., 1991, for a review). There are several types of OAEs, but spontaneous otoacoustic emissions (SOAEs) are especially interesting. SOAEs are essentially tonal sounds that are continuously emitted at low sound-pressure level (SPL). These emissions are quite stable in frequency, but can vary considerably in level in response to various influences (Probst et al., 1991). While about 60-70% of all people with normal hearing have one or more SOAEs (e.g., Talmadge et al., 1993), they typi- Corresponding author. Fax: (512) cally pass unheard by their owners, presumably due to some form of long-term perceptual adaptation. SOAEs are not common in mammals below the highest primates, even though other forms of OAEs are (Probst et al., 1991). Hearing is slightly more sensitive in ears having several SOAEs than in ears having none (Mc- Fadden and Mishra, 1993). The most common form of SOAE is not found in frequency regions exhibiting hearing losses greater than about 30 db [although a relatively rare form of high-amplitude SOAE can occasionally be found in ears with hearing loss (Probst et al., 1991)]. Accordingly, the existence of SOAEs is widely regarded to require an essentially normal cochlea and, more specifically, a normal complement of outer hair cells. One way to think about SOAEs is that they result from a localized anomaly of some sort along an otherwise-regular cochlear partition; however, the physiological basis for SOAEs has yet to be established with certainty. SOAEs and other forms of otoacoustic emissions have been the object of considerable Elsevier Science B.V. SSDI (95)

2 182 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) research interest in recent years because of their potential to provide information about cochlear mechanics non-invasively (Probst et al., 1991). The prevalence of SOAEs is similar in infants and adults (Strickland et ai., 1985; Burns et al., 1992; Bonfils et al., 1992). While long-term longitudinal studies on SOAEs have yet to be conducted, various labs have reported only small percentage changes in the acoustic frequencies of SOAEs in adult ears that have been repeatedly tested over the course of the approximately 15 years since SOAEs have been discovered (Probst et al., 1991; Burns et al., 1993; but compare K6hler and Fritze, 1992). Also, Burns et al. (1994) found only minor fluctuations in the acoustic frequencies of SOAEs monitored in children over the first two years of life. Thus, the available evidence supports the assumption that SOAEs are a reasonably stable trait through life--with the qualification that SOAEs can be lost when the relevant outer hair cells are damaged and hearing is lost, either permanently or temporarily (e.g., McFadden and Plattsmier, 1984; Long and Tubis, 1988; McFadden and Pasanen, 1994; Norton et al., 1989). SOAEs are more common in females and right ears than in males and left ears (reviewed by Bilger et al., 1990; also see Burns et al., 1992; McFadden, 1993a and McFadden, 1993b; Penner et al., 1993; Talmadge et al., 1993; Whitehead et al., 1993). Bilger et al. (1990) used the sex difference in SOAEs to argue that the tendency to express SOAEs may be a dominant X-linked trait. Additional support for the possibility of a genetic contribution to the expression of SOAEs came from the discovery that SOAEs are more common in Negroes and Asians than in Caucasians having light eyes (Whitehead et al., 1993), and from preliminary reports that the number of SOAEs exhibited is more similar in monozygotic (MZ) co-twins than in dizygotic (DZ) cotwins (Russell and Bilger, 1992; Sirianni et al., 1992). In addition, the existence of strong prenatal contributions to the expression of SOAEs was implied by the finding that females with male co-twins have relatively few SOAEs, and are, in this regard, more similar to males than to other females (McFadden, 1993b; for a review of related effects, see Miller, 1994). The possible contribution of genetics to SOAEs is considered further in this paper. A previous report (McFadden, 1993b) utilized data from the current experiment, but the presentation and analyses were substantially different because a different scientific point was at issue. The present paper also provides a more complete description of the methods than was possible in the previous report. At points in this paper, we will compare our results with those of Russell (1992), whose twins experiment became known to us only after ours was well along. Russe l measured SOAEs in 60 pairs of twins--30 male and 30 female pairs, equally divided between MZ and same-sex DZ (SSDZ), sample sizes that are roughly comparable to ours. 2. Methods 2.1. Subjects Subjects were recruited by obtaining from our university registrar a list of students having the same last name and birthdate. Letters describing the experiment were sent to likely prospects, and this was followed up by a telephone call. Prospective subjects were asked whether they were twins, and when the answer was yes, the three first-level questions from Nichols and Bilbro (1966) were asked in an attempt to determine whether they were identical or fraternal twins. Initial testing focussed on likely identical twins, with same-sex fraternals and non-twins being added once it was clear that a complete experiment was worthwhile. Still later, opposite-sex twins (OSDZs) were recruited and tested. The non-twins were recruited by word of mouth and through notices published in the student newspaper and distributed to large undergraduate classes. In accord with common experience (Lykken et al., 1978), DZ and male twins were generally more difficult to recruit than MZ and female twins, so in the later stages of the experiment, recruitment focussed on these groups and was expanded to include advertisements in high school and regional newspapers. The twins tested early in the experiment were paid $20 each for the two-hour test session; this was later increased to $40 each when locating and recruiting additional sets of twins became increasingly difficult. Non-twins were paid $15 each for the two-hour session. As a group, OSDZ twins of college age were at least as difficult to recruit as SSDZ males, often because the OSDZ males expressed attitudes ranging from ambivalence to outright hostility towards an experiment on twins. By the end of the approximately one year of data collection, it was evident that recruitment of additional twins in this geographic area had become a matter of rapidly diminishing returns. A tally of the subjects lost to data analysis for various reasons is presented in the Results section Procedure During the two-hour test session, all subjects independently completed a questionnaire that included items on personal characteristics such as height and weight, eye and hair color, handedness and footedness, use of prescription drugs, prematurity of birth, etc. Also included were a set of questions suggested by Nichols and Bilbro (1966) for determining zygosity. The two-level scoring procedure developed by Nichols and Bilbro (1966) was used with one exception; when

3 D. McFadden, J. C. Loehlin / Hearing Research 85 (1995) their second-level rules did not produce an unambiguous categorization for a set of twins, no 'intuitive judgment' was made. Rather, the data from those subjects were excluded from all analyses and presentations. In general, questionnaire procedures of this sort correctly identify the zygosity of twins well over 90% of the time (Plomin et al., 1990, p. 315). Subjects were dichotomized into dark- and light-eyed using the following procedure. The left eye of each subject was matched to one of a series of 12 artificial eyes manufactured by the American Optical Company, covering the range from very dark brown to light blue. The spectral characteristics of the irises of these artificial eyes were determined using a spectroradiometer (Photo Research, model PR-704) under the same fluorescent illumination as present in the test room. The six artificial eyes characterized as dark were well separated in the C.I.E. coordinate system from those characterized as light, primarily with respect to the u' dimension, which roughly indicates the relative proportions of the red and green primaries. The dark eyes all had u' values greater than 0.274, with v' values ranging from to The six eyes characterized as light all had u' values less than 0.234, with v' values ranging from to (for details of these units see Wyszecki and Stiles, 1982). A portable screening audiometer (Maico MA40) was used to test the hearing of all subjects, but the emissions measurements were made irrespective of performance on this test. Later, emissions data were discarded for all subjects whose hearing was not within 20 db of normal over the frequency range of 1000 to 4000 Hz. For the measurement of SOAEs, two co-twins (or two non-twins) were simultaneously placed in different sound-proofed rooms equipped with slightly different systems for recording emissions. The subjects lay on small camp cots with pillows supporting the electrical and acoustical leads coming from the probe assembly inserted into the external ear. A continuously transmitting intercom allowed the subjects to communicate with the experimenter as desired. Because various demonstrations have revealed the importance of a period of relaxation prior to the measurement of SOAEs (Zurek, 1981; Whitehead, 1991; McFadden and Pasanen, 1994), all of our subjects reclined on their cots in the quiet for at least 15 minutes prior to data collection. In addition to SOAEs, click-evoked OAEs were also obtained during these sessions, along with certain psychophysical measures; those data will be reported elsewhere. SOAEs were measured using Etymotic low-noise microphone systems. In one test room, an ER-10 insert microphone was followed by an ER10-72 pre-amplifier, and in the second test room, an ER10B insert microphone and pre-amplifier system was used. The output of each pre-amplifier was passed through a custombuilt, low-noise amplifier/filter combination that provided about 30 db of fixed gain and high-passed the waveform at 400 Hz. Those fixed gains were adjusted so that the two recording systems provided the same overall sensitivity for a 1000-Hz signal of moderate level. Following this adjustment, one of the two total systems was more sensitive in some frequency regions, and the other more sensitive in other frequency regions, but the two systems were always within 1 db of each other over the frequency range of Hz, and within 2 db at 5000 Hz, for signal levels well above the noise floor. The electronic noise floors in 5-Hz bands centered at 1000 and 2000 Hz were about db and -6.0 db SPL, respectively, for the ER-10 system, and about -0.5 and -3.5 db SPL, respectively, for the ER-10B system with the microphones placed in small calibration cavities. The same recording system was used for both ears of a subject. The output of each amplifier/filter was led to a Nicolet/Wavetek 444a Mini-Ubiquitous spectrum analyzer located outside the test rooms. The analyzer was set to sum eight consecutive 'samples' (Fast Fourier Transforms or FFTs). FFTs were collected for both the 2-kHz and 5-kHz low-pass ranges for each ear studied; the frequency resolution was 5 and 12.5 Hz, respectively, for these two frequency ranges. For those spectral peaks that were easily identified visually in either of these FFTs, an additional averaged FFT was collected for a 100-Hz window surrounding the peak. All FFTs were saved as data files for subsequent analysis. The probe assemblies inserted in the ear canals also contained a small plastic tube attached to a miniature earphone that was used to present clicks for eliciting another type of otoacoustic emission; the results of those measurements will be presented elsewhere. Spectral peaks were identified in the 2-kHz and 5-kHz data files using an objective algorithm that successively compared the outputs of a sliding wide-band window (60 or 150 Hz wide, respectively) with a sliding narrow-band window (20 or 50 Hz wide, respectively) centered within it. The algorithm characterized as an SOAE any positive difference of 2 db or more; for many subjects a 2-dB peak amounted to about four standard deviations above the background variability. On rare occasions the decision of the objective algorithm was overridden when supplementary information from a 100-Hz FFT was contradictory, but the latter were never used to determine the frequency or the level of an SOAE. All SOAEs were identified by the first author. SOAE identification was accomplished without information about the zygosity of the subjects, and zygosity was determined in ignorance of the SOAE results. For several reasons, no attempt was made to compensate for the small differences in the noise floors of

4 184 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) the two recording systems used. First, the level of the SOAEs was not of major interest here--the number and acoustic frequencies of the SOAEs were. Second, in our experience, the background noise out of which SOAEs are extracted is not established by the noise floor of the electronics, but by the ambient body noise generated by the individual subject. In this study, the latter was typically 2-4 db greater than our electronic noise floor and was highly variable within and across subjects, meaning that the 1-3 db difference in the noise floor between our two recording systems played a minor role in our ability to detect SOAEs. Because more elaborate, off-line procedures exist for extracting SOAEs weaker than those we could detect (e.g., Bilger et al., 1990, p. 427; Russell, 1992; Penner et al., 1993; Talmadge et al., 1993), our results should be viewed as conservative; additional, weak SOAEs surely were present, but undetected. Although the sound-pressure level of each detected SOAE was recorded, those measures were not used in the analyses reported here. SOAE level is known to be sensitive to a number of variables, and measurements of SOAE level can vary considerably within and across sessions (e.g., Whitehead, 1991; McFadden and Pasanen, 1994). By comparison, SOAE frequency is generally quite stable. 3. Results 3.1. Attrition Data are reported for 242 subjects. The data for a number of additional subjects were discarded for various reasons. Data for five sets of twins were lost because zygosity could not be unambiguously determined using the Nichols and Bilbro procedures described above (those procedures, originally developed for high-school-age twins, had particular difficulty classifying pre-teen twins). Hearing sensitivity not within 20 db of normal on the audiometric screening test for one or both co-twins eliminated two sets of MZs and two sets of OSDZs, as well as one male OSDZ and two male non-twins. Technical or equipment problems eliminated one set of twins and two non-twins. One male/female pair was omitted because they had been born triplets. One OSDZ female was eliminated as an outlier because her 6 SOAEs in each ear put her 7.8 standard deviations above the mean of the rest of her group. Were this subject's data not excluded, the mean number of SOAEs per ear and the mean per emitting ear (Table 2) for OSDZ females would be 0.97 and 1.94, respectively. However, her exclusion is irrelevant Table 1 Numbers (and, in italics, proportions) of subjects of differing types having No SOAEs, SOAEs in one ear only, or SOAEs in both ears SOAEs Monozygotics Same-Sex Dizygotics Non-Twins Opposite-Sex Dizygotics Dark-Eyed None Left Ear Only 1 0.O4 Right Ear Only Both Ears Total Subjects % Dark-Eyed 0.52 Mean Age (years) d All 9 d All ~ d All 9 d All , Light-Eyed None Left Ear Only Right Ear Only Both Ears Total Subjects % Light-Eyed 0.48 Mean Age (years) t l , ,2 19.6

5 1.00 D. McFadden, J. C. Loehlin / Hearing Research 85 (1995) for the primary analyses in this paper, which focus on same-sex twins Distribution of SOAEs across sex and ear The basic results are summarized in Tables 1 and 2, where the data for the dark-eyed and light-eyed subjects are reported separately. In Table 1, the prevalence and distribution of SOAEs are shown for the various twin and non-twin groups. For this table, the entries are the number (and proportion) of subjects having the combination of characteristics shown. Also shown are the number and average ages of subjects in the different groups and the percentages of each that were dark- and light-eyed. In Table 2 are shown the total number of individual SOAEs detected in the different types of subjects, along with the Mean (numbers of) SOAEs per Ear, the Mean (numbers of) SOAEs per Emitting Ear, and the Median (numbers of) SOAEs for All Ears and for Emitting Ears only. Included, and appropriately categorized, in Tables 1 and 2 are four female and two male SSDZ pairs in which one co-twin was dark-eyed and the other lighteyed. Prior to presenting detailed analyses of the data, we shall note several interesting features of the basic results shown in Tables 1 and 2. Perhaps the most striking features of these data are the sex and ear differences. In every one of the eight subject-type by eye-color groups in Table 1, the proportion of females who exhibited SOAEs was greater than the proportion of males who did (i.e., the 'None' proportions are always greater for the males). And, for the sexes combined, the proportion of subjects in each of the eight subgroups exhibiting SOAEs in the right ear was greater than the proportion exhibiting SOAEs in the left. Both of these findings are in line with previous reports (e.g., Bilger et al., 1990; Burns et al., 1992; Russell, 1992; Whitehead et al., 1992; Penner et al., 1993; Talmadge et al., 1993). Considering just our non-twins, 63% of the females and 47% of the males had at least one SOAE. In the Bilger et al. (1990) meta-analysis, the corresponding (non-twin) numbers were 53% and 27% for females and males, respectively, and in recent surveys, conducted with more sensitive measurement procedures, the prevalences have been about 75-85% for females and 45-65% for males (Burns et al., 1992; Whitehead et al., 1992; Penner et al., 1993; Talmadge et al., 1993). Not only did sex and ear differences exist with regard to the presence or absence of SOAEs, they were also evident in the number of SOAEs exhibited in those subjects having SOAEs. In Table 2, the Mean (number of) SOAEs per Emitting Ear is greater for females than for males in each of the four same-sex twin groups (the non-twins showed the trend for darkeyed individuals only, and the OSDZs appear to consti- Table 2 Numbers (and, in italics, proportions) of SOAEs in the two ears of the different types of subjects SOAEs Monozygotics Same-Sex Dizygotics Non-Twins Opposite-Sex Dizygotics <2 <5 All <2 <5 All <2 ~ All ~ <5 All Dark-Eyed Left Ears Right Ears Total SOAEs Mean SOAEs per Ear Standard Deviation a Median for All Ears Mean per Emitting Ear Median for Emitting Ears Light-Eyed Left Ears Right Ears Total SOAEs Mean SOAEs per Ear Standard Deviation ~ Median for All Ears Mean per Emitting Ear Median for Emitting Ears a Standard deviations for SOAEs per Ear in preceding row

6 186 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) tute a special caseisee McFadden, 1993b). The mean number of SOAEs for individuals who emitted SOAEs was greater for right than for left ears in seven of the eight subgroups. The exception was the light-eyed SS- DZs, but the numbers there are small--the means were based on only 4 left ears and 6 right ears in this group. These trends suggest that the presence or absence of SOAEs and the number of SOAEs among those who display them are both reflections of a single common mechanism--a continuously varying tendency to produce SOAEs. The overall measure that seems best to capture both the presence/absence of SOAEs and their number when present is the Mean number of SOAEs per Ear, taken over all individuals in a group. These means are shown in Table 2 and displayed graphically in Fig. 1. [The data in Tables 1 and 2 and Fig. 1 are the same as in the correspondingly numbered, but less detailed, tables and figure of McFadden (1993b).] The distributions of SOAEs are quite skewed, as evidenced by the fact that the standard deviations in Table 2 often exceed the means. A logarithmic transformation (see below) was used to reduce the skewness for purposes of statistical analyses. Further, Medians are given in Table 2 for SOAEs across All Ears and across Emitting Ears only. Although the medians are rather lacking in resolution, they typically agree with the means, e.g., in being higher for females. In addition to the sex difference previously discussed, Fig. 1 suggests a relative excess of SOAEs among MZ twins, particularly MZ females, and among dark-eyed relative to light-eyed subjects. Neither of these trends in our data was unequivocally supported by the statistical analyses to be reported, but a similar direction of MZ-DZ difference is also present in the data of Russell (1992), and the eye-color difference may be related to the higher prevalence of SOAEs in non-caucasians reported by others (Russell, 1992; Whitehead et al., 1993). The striking paucity of SOAEs in OSDZ females was the subject of a separate report (McFadden, 1993b). The various similarities between our data and Russell's (1992) are encouraging because there were a number of procedural differences in the two studies--perhaps most importantly, her use of off-line processing allowed her to detect, on the average, about twice the number of SOAEs that we detected. In order to test for differences across subject groups and conditions, a four-factor analysis of variance was conducted using as the dependent variable a logarithmic transformation of the number of SOAEs (see next paragraph). The factors were Subject Type (MZ, SSDZ, and Non-Twin), Eye Color (Dark and Light), Sex, and Ear of Measurement (Left and Right), with the last factor having repeated measures. The main effect for sex was significant, F(1,134)= 14.72, p = , as was that for ear of measurement, F(1,134)= 17.58, p < Not statistically significant were the main effects for Subject Type, F(2,134) = 2.43, p = 0.092, or Eye Color, F(1,134)= 2.91, p = Of the various DARK-EYED SUBJECTS rr 2.0 ~ 21,,,<,5 24 Wz~/ //, / / '//S~///// < o.~ i ' U / ~ /[/[/[///// Ii O 3.0 1:22] FEMALES Pl- ~ MALES o #Y//~;: = < 1.5 ~///z//,: ///////. ~////,4. N LIGHT-EYED SUBJECTS Same-Sex Opposite-Sex MONOZYGOTICS DIZYGOTICS NON-TWINS DIZYGOTICS Fig. 1. Mean number of SOAEs per ear shown for the different subject groups, sexes, and eye colors. The number of subjects contributing to a condition is indicated above each bar.

7 D. McFadden, J.C. Loehlin / Hearing Research 85 (1995) interactions, only that for Ear X Sex even approached significance, F(1,134)= 2.54, p = [This ANOVA was implemented with SYSTAT MGLH, a program that uses least-squares estimates to deal with unequal numbers of subjects in groups--see SYSTAT (1992).] The preceding analysis was conducted on a logarithmic transformation of the scores to decrease skewness. The transformation was y = ln(x + 1), where x is the number of SOAEs, In is a natural logarithm, and the 1 was added to avoid zeroes. This transform does not produce a strictly normal distribution (the excess number of zero scores precludes that), but it does serve to reduce the impact of individuals with many SOAEs. Throughout this paper we will use the logarithmically transformed scores for all statistical analyses and modeling, and the non-transformed scores for descriptive presentations. For the ANOVA described, care was taken to respect the assumption of independence of scores (one would not wish to assume that the scores of two co-twins would be independent of one another, particularly for the MZs). Specifically, each twin pair --with the exceptions noted below--contributed only one score to the various conditions in the analysis; each score was a two-ear average of the number of SOAEs exhibited in the left (or right) ears of the pair of co-twins. In the case of the four sets of female and two sets of male SSDZ twins that had opposite eye colors, each co-twin contributed a one-ear score to the appropriate eye-color group. Of course, one-ear scores were also used for individual non-twins. OSDZ twins were excluded from this analysis because OSDZ females have been shown to exhibit fewer SOAEs than other females (McFadden, 1993b), and their inclusion could have distorted the analysis. For comparison, the twins data of Russell (1992) were similarly transformed and analyzed. The main effects for Sex and Ear were likewise significant for those data, as was Race (Euro-Americans and Afro- Americans). The main effect for Twin Type (MZ vs. SSDZ) and all interactions were not Heritability estimates If the number of SOAEs is influenced by the genes, one expects MZ co-twins, who are genetically identical, to be more similar than DZ co-twins, who share, on the average, only one-half their genes. Were DZ co-twins found to be about as similar as MZ co-twins, one would suspect that some shared factor or factors in the environment to which the twins have been exposed was accounting for the resemblance in the number of SOAEs exhibited, and that the genes were not making any marked contribution to individual differences. (Of course, the genes still would presumably be contributing to the construction of the physiological mechanisms of the inner ear- just not to the differences in them Table 3 Correlations of number of SOAEs for monozygotic (MZ) and samesex dizygotic (SSDZ) twins Correlation Present Study Data of Russell MZ SSDZ MZ SSDZ Across twins, corresponding ears Across twins, non-corresp, ears Two ears of same person Number of twin pairs Intraclass correlations (double-entry Pearsons). Sexes, eye colors, and ears pooled. Within-person correlations based on all individuals in category, that underlie the variation in SOAE expression.) In order to describe the relative similarities between MZ and SSDZ co-twins, correlations were calculated for various combinations of ears using the non-transformed number of SOAEs. Table 3 contains intraclass correlation coefficients for MZ and SSDZ pairs both within and across co-twins. Correlations for pairs are given for corresponding and non-corresponding ears, where corresponding refers to, e.g., the left ear of one co-twin paired with the left ear of the other co-twin, and non-corresponding refers to the left ear of one co-twin paired with the right ear of the other. Both co-twins of a pair were used in obtaining the correlation between left and right ears of the same person. The various correlations were obtained after pooling over sex, eye color, and, where possible, ears, to achieve reasonable sample sizes. For comparison, similar calculations made from the Russell (1992) data are also shown. The principal message of Table 3 is clear: MZ co-twins yield much higher correlations than SSDZ twins--about equally so for corresponding and noncorresponding ears. One striking feature of the correlations in Table 3 is that, in both data sets, the three MZ correlations are quite similar: so far as number of SOAEs is concerned, an ear of an MZ twin resembles the same ear or the other ear of his co-twin about as much as it does the ear on the other side of his own head. A process that is basically driven by the genes with some amount of random error in the development and/or measurement of the trait would have this characteristic; it is difficult to imagine an environmentallydriven process that would. Whitehead et al. (1993) made a similar comparison of number of SOAEs in the two ears of the same subjects and obtained correlation coefficients of about 0.59 and 0.53 for non-twin females and males, respectively; the corresponding values for our non-twin females and males were 0.60 and [There is also evidence that the patterns of click-evoked emissions are similar in the two ears of normal-hearing non-twins (e.g., Probst et al., 1986).] Note that one would not expect the correlations across the ears of the same

8 188 D. McFadden, J.C. Loehlin / Hearing Research 85 (1995) individuals to differ systematically across groups in Table 3, except perhaps via indirect effects, such as sex, eye color, or the like, for which our twin groups do show some differences. Also note that the correlations for the SSDZ twins in the Russell experiment were larger than those for our SSDZs, and were about half the value of the correlations for her MZ twins, as would be expected if the genes affecting SOAEs were largely additive in their action. Further, the great similarity in the MZ correlations across the two experiments supports our earlier contention that the small differences in the noise floors of our two recording systems could be safely ignored. We can go beyond simple qualitative statements by fitting an explicit model to the data, which will also facilitate our asking such questions as whether the ears or the sexes differ with respect to the influence of the genes on individual differences in SOAEs. (This is, of course, a different question from whether such variables have effects on mean numbers of SOAEs, a question that has already been clearly answered in the affirmative.) The parameters of such model-fitting will also give us quantitative estimates of the heritability of SOAEs; that is, the proportion of their variance that is associated with genetic differences among the individuals of the population in question. Fig. 2 presents a model of the situation in the form of a path diagram. Following the usual conventions of latent-variable analysis (Loehlin, 1992a), the squares at h~ MZ = 1.0 ~...I~ = 0.5 TWIN A C~ 1,0 h~ c~ ' -Jl e~ s e: TWIN B Fig. 2. Path diagram for analysis of SOAE correlations shown in Table 4. Observed variables (in squares): A i. = left ear of co-twin A, etc. Latent variables (in circles): G A genes of co-twin A; C A = twins' shared environment for co-twin A; etc. Paths: h = additive effect of genes; c = effect of shared environment; e ~ residual effects (nonshared environment, error); subscripts d and e = left and right ears; s = correlated residual effects across the ears of an individual. The genetic correlation between twins (top left of the figure) is 1.0 for MZ twins and 0.5 for SSDZ twins; all other parts of the figure are assumed the same for MZ and SSDZ twins. Table 4 Correlations of log number of SOAEs for left female and male MZ and SSDZ twins and right ears of Group Pairs Across twins Same person LALB RARB LAR~ RALB LARA LBRB MZC MZd SSDZ? SSDZd Single-entry Pearson correlations estimated for censored distributions (see text). L, R = left, right ear; A, B = first and second co-twin of a pair. The score transformation used was ln(x + 1), where In is a natural logarithm, and x is the number of SOAEs. Entries under LL and RR are for corresponding ears; entries under LR and RL are for non-corresponding ears. the bottom of the figure represent the measurements for a typical pair of co-twins--the number of SOAEs in the left and right ears of each member of the twin pair. In each case, three sets of possible influences are represented: the genes, via paths h; the shared environment of the twins, via paths c; and a residual, e, which would encompass random environmental effects and errors of measurement. At the top of the figure, the curved arrows represent correlations: the shared environments of the twins are correlated 1.0, by definition; the twins' genotypes are correlated 1.0 if they are MZs, and 0.5 if they are DZs. This assumes a simple additive genetic model and mating that is not based on the number of SOAEs exhibited by potential mates (non-assortative mating). Near the bottom of the figure are curved arrows labeled s, which allow for the possibility of environmental factors which affect the two ears of a given individual, but not his or her co-twin. This additive model assumes that broad- and narrowsense heritabilities are the same (e.g., Plomin et al., 1990); accordingly, we simply use 'heritability' throughout. Non-additive effects are discussed below. Table 4 presents the data to be fitted. Correlations are shown for various combinations of male and female MZ and SSDZ twins' left and right ears, within individuals and across pairs. These are single-entry Pearson correlations based on pair members arbitrarily designated as twin A and twin B, rather than the intraclass correlations which were presented in Table 3 for descriptive purposes. Correlations (or covariances) of this type are commonly used for model fitting with twin data (Neale and Cardon, 1992). Because the significance tests are sensitive to skewness in the underlying distributions, the logarithmically transformed scores described earlier were used for these correlations. Also, because these distributions can be viewed as 'censored,' i.e., as reflecting an underlying continuous distribution with a threshold below which all scores are zero, the correlations in Table 4 were estimated using the pro-

9 D. McFadden, J.C. Loehlin / Hearing Research 85 (1995) gram PRELIS (SPSS Inc., 1990), which produces maximum likelihood estimates of true correlations for such variables. A Monte Carlo study has shown that if ordinary correlations are used with censored variables, genetic model fitting can produce inflated chi-squares and distortion of heritability estimates (Waller and Muth~n, 1992). The correlations in Table 4 are generally higher for MZ twins than for SSDZ twins, just as they were in Table 3. Also notable are some anomalous featuresnamely, the correlations for the two ears of the same person are lower for the SSDZs than for the MZs, and the correlations for SSDZ females are markedly lower than those for SSDZ males, a pattern that does not exist for the MZ twins. An outcome of the latter sort suggests additive genetic effects for males and genetic dominance for females, but complex models of this sort are not explored further here, in part because the data of Russell (1992) suggest simple additive genetic effects for both sexes. Models were fitted to the correlation matrices whose off-diagonal elements are given in the rows of Table 4, using the program LISREL with a maximum-likelihood criterion (J6reskog and S6rbom, 1989). This program yields estimates of the values of the various paths in Fig. 2 at the point of best fit of a given model, plus an approximate chi-square for the goodness of that fit. If the chi-square is statistically significant, it means that the model does not fit the data, and can be rejected. If the chi-square is not significant, the model is retained as consistent with the data. Differences in the fits of nested models (where one is a subset of the other) can be appraised by the difference in their chi-squares because that difference is itself a chi-square with degrees of freedom equal to the difference in dfs of the models being compared. It is customary in such model fitting to retain the simplest model that adequately fits the data. While we will follow this practice, we will sometimes discuss effects which were not large enough to achieve statistical significance in our data, but are consistent with effects observed by others. All the statistical results to be reported should be interpreted as at best approximate, for at least three reasons: (1) the statistics assume multivariate normality, and the normality of SOAEs must remain in question even after log transformation and the allowance for censored distributions; (2) the statistical theory is based on covariances, and we are using correlations, adjusted for df in the manner recommended by Neale and Cardon (1992); and (3) the statistical theory is a large-sample theory, and our samples are small. Some model-fitting results are presented in Table 5. The first row shows the full model represented in Fig. 2, with parameters equated for males and females. The chi-square, relative to its degrees of freedom, suggests Table 5 Fits of simple heredity-environment models to correlations in Table 4 Model X 2 df p Xd2iff df p from line 1. hl, h r,cl,cr,s, <0.05 el, er 2. h, e > > 0.90 (1) 3. hf, hm, el, e m > > 0.10 (2) that this model does not represent a satisfactory fit to the data. The second row shows a much simpler model, one which equates the h and e parameters across ears and sexes and sets the shared-environment parameters c and s to zero. This model is not rejected by the data. The model in the third row is like the second, but with h and e permitted to differ for the two sexes. This model is also acceptable, but it is not significantly better than the simplest model. Several points are worth noting. First, none of these models fits the data exceptionally well, although two of the three are statistically acceptable, as shown by the non-significant chisquares. Second, the simple two-parameter model fits almost as well in absolute terms as the full model, despite five fewer parameters solved for. Third, allowing for sex differences (row 3) results in a modest improvement in the fit, although not a statistically significant one. The somewhat marginal fits of the models can be attributed in part to the previously noted irregularities in the data that were evident in the correlations in Table 4--namely, the SSDZ females appear to show less resemblance across co-twins than do the SSDZ males, and the MZs appear to show more resemblance between the ears of individuals than do the SSDZs. While these anomalies may arise merely from sampling error, they necessarily lead to caution when interpreting the results. Nevertheless, because the present estimates agree reasonably well with estimates derived from the twins data of Russell (see below), we consider them to be creditable. In Table 6 are shown the heritability estimates from the three models of the preceding table (they are simply the squares of the estimate of path h in each case). They represent the best estimate, given the model being fitted, of the proportion of the variance of this trait in this population that is due to the genes. Also shown, for comparison, are estimates calculated using the same procedures from the data of Russell (1992). Table 6 Heritability estimates for models in Table 5 Model Present Study Data of Russell 1. Full model h 2 = 0.69, h2r = 0.68 h 2 = 0.77, h2r = One h, one e h 2 = 0.71 h 2 = Sex differences h 2 = 0.80, h 2, = 0.60 h 2 = 0.83, h2~ = 0.72

10 190 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) For Russell's data, all three Table 5 models fit acceptably well (ps of > 0.10 to > 0.30). As in the present study, neither the row 1 model nor the row 3 model represents a statistically significant improvement over the simplest model, although both data sets show a trend for the heritability to be higher for females. [The absence of non-additivity in the Russell data convinced us that the hint of non-additivity in our data (Table 4) did not warrant the testing of non-additive genetic models.] Russell herself used a different approach to heritability analysis that yielded somewhat different estimates from those in Table 6, but her analyses led to the same conclusion--genes are responsible for the bulk of individual variation in SOAE expression. Thus, the simplest tenable interpretation of all the data would seem to be that somewhere around threequarters of the individual variation in the tendency to exhibit SOAEs is due to the genes (perhaps a little more in females than in males) and that the remainder is due to factors that are unshared between twins or even between the two sides of an individual's head (presumably small-scale local incidents during development, and errors in the measurement process). The possibility of higher heritability in females is interesting because it suggests that the basic gene-influenced physiological process is better expressed in females, perhaps because, for some reason, the process is subject to greater interference in males than in females. The preceding analysis assumed a DZ genetic correlation of Actually, DZ twins can share slightly more than one-half of their genes if their parents tend to be similar on a trait. There is, of course, no reason to suppose that spouses directly select one another on the basis of their individual tendencies toward emitting weak sounds from their inner ears- of which both are unaware--but secondary associations, such as ethnicity, might result in a small correlation between partners. As a check on the consequences of this possibility, the 2-parameter model shown in the second row of Table 5 was re-fitted with the genetic DZ correlation increased to This would be equivalent to a quite substantial degree of correlation between spouses on a trait or traits moderately related to SOAEs. The fit of the model was essentially the same (X 2 of versus 34.79), as was the heritability estimate (0.72 versus 0.71). Because this leads to no real change in interpretation, the possible presence of a modest correlation between spouses can safely be ignored Supplementary observations An informal examination of the SOAE data revealed no obvious contribution from prematurity of birth and/or birth weight. Most twins are born prematurely (Bulmer, 1970), but the SOAEs of premature non-twins appeared no different from those of full-term non-twins. Also, handedness and footedness (Mac- Neilage, 1991) seemed to be unrelated to the expression of SOAEs. The left-footed subjects, both twins and non-twins, had more SOAEs in the right ear than the left, just like the right-footed subjects, and the number of SOAEs per ear in left-footers ranged from zero to 9. Four sets of MZ twins were of opposite handedness (and footedness), which is a primary characteristic of mirror-image twins (Bulmer, 1970). Two of these sets were male and exhibited no SOAEs. The other two sets were female. Of the latter, one set did appear to have greater similarity in the acoustic frequencies of their SOAEs for non-corresponding ears than for corresponding ears, but the other set did not. Accordingly, no special treatment was given to the data from these two sets of possible mirror-pairs in any of the analyses reported here. A few SOAEs fell at or near frequency values corresponding to the two distortion products (fhigher--fl... ) and (2f fhighcr ) potentially generated by other SOAEs in that ear. No attempt was made to exclude these potential distortion products from our data set prior to analysis. One reason was that, early in the experiment, many of the apparent correspondences with a predicted distortion-product frequency seen with the 5-Hz spectral resolution that was used for detecting SOAEs below 2000 Hz were found not to be actual correspondences when expansion files having 0.25-Hz resolution were examined--and collecting data with 0.25-Hz resolution was much too inefficient for routine use. Second, time did not allow tests (such as suppression of one SOAE by an external tone) typically used to verify that an SOAE is the product of two other SOAEs acting as primary tones (Burns et al., 1984). To our knowledge, no previous study on the prevalence of SOAEs has excluded possible distortion products either. 4. Discussion As an aid to the reader not knowledgeable about the physiology of hearing or the details of SOAEs, we will begin this discussion by presenting a working hypothesis about the origin of SOAEs that is consistent with the primary facts available. As the discussion proceeds, the evidence and interpretations that led us to advance this hypothesis over other possibilities will emerge. The available evidence suggests to us that, at the outset, most embryos (ignoring those with a genetic auditory malady) have the potential to exhibit relatively large numbers of SOAEs postnatally. Operating against this potential to exhibit large numbers of SOAEs are a number (unknown) of perfectly normal, pre- and post-

11 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) natal processes that act to diminish the number of potential SOAEs. Among these is the process of masculinization, which reduces the prevalence of SOAEs in males and (indirectly) in OSDZ females (McFadden, 1993b) by eliminating potential SOAEs--perhaps at random, but certainly in no obviously systematic way. For some reason, one or more of these prenatal processes may act less effectively on MZ twins, especially MZ females, than on other fetuses, leaving them with a relative abundance of SOAEs. According to this view, then, only those SOAEs that escape a hypothesized process of elimination are expressed. Attrition explanations of this sort have the virtue that the high prevalence of SOAEs in females generally, and in MZ females in particular, is attributed to a failure of SOAEs to be eliminated rather than to extra SOAEs being added. Further, under accounts based upon differential attrition, the appearance of heritability being lower in males and left ears than in females and right ears (Table 6 above) can be attributed to more SOAEs being randomly eliminated in males and left ears than in females and right ears. Thus, SOAEs in female and right ears may provide a clearer window onto the underlying genetics than do the SOAEs in male and left ears. Also suggested as possible factors contributing to sex, ear, and age differences in SOAEs are differences in the outer/middle ear system and in the efferent supply (Previc, 1991; Burns et ai., 1994; McFadden, 1993a) Possible physiological bases for SOAEs It is important to be clear about exactly what is being inherited. An SOAE, in and of itself, surely has no evolutionary value. Rather, it should be thought of as epiphenomenal to a trait that presumably was under selection pressure, such as hearing sensitivity in the quiet. Current belief is that the first db of auditory sensitivity requires a contribution from an array of metabolically dependent 'cochlear amplifiers' that act to increase locally the displacement of the cochlear partition at low sound-pressure levels (Davis, 1983). The physiological mechanisms constituting these narrowband cochlear amplifiers remain unknown, but the presence locally of intact outer hair cells is known to be crucial. SOAEs may originate from small variations in the strength of the cochlear amplifiers located at a particular location (frequency region) along the cochlear partition, or from anomalies of other sorts in those cochlear amplifiers. Since the hearing of people having several SOAEs is somewhat more sensitive than that of people having no measurable SOAEs, even in frequency regions distant from the SOAEs (McFadden and Mishra, 1993), the presence of SOAEs is widely regarded to be emblematic of excellent hearing, and consequently, of (otherwise) highly effective cochlear amplifiers. ~ Apparently, evolutionary pressure to produce highly sensitive hearing led to the mechanisms underlying the array of cochlear amplifiers, and SOAEs are relatively innocuous 'natural imperfections' (Lonsbury-Martin et al., 1988) that occasionally result. Several suggestions have been made about the possible nature of the anomaly in cochlear mechanics that gives rise to an SOAE. Early in the study of otoacoustic emissions, it was repeatedly suggested that SOAEs might be the result of localized damage to the organ of Corti, perhaps like that seen following exposure to intense sounds (Ruggero et al., 1983; Clark et al., 1984; Zurek and Clark, 1981). This explanation of SOAEs has become increasingly unlikely over the years [with the possible exception of the high-frequency, high-level SOAEs that are occasionally seen (Probst et al., 1991)]. The counterevidence includes the facts that SOAEs have about the same prevalence in infants as in adults (Strickland et al., 1985; Burns et al., 1992; Bonfils et al., 1992) even though the two have markedly different histories of noise exposure, the number of SOAEs does not increase with age (Strickland et al., 1985; Burns et al., 1993,1994) even though other consequences of noise exposure do, SOAEs are more prevalent in females than males (e.g., Bilger et al., 1990; present data) even though males are generally exposed more to intense sounds than females (e.g., Axelsson et al., 1987), and ears exhibiting several SOAEs have better, not worse, hearing than ears exhibiting none (McFadden and Mishra, 1993). Also of importance was the demonstration by Lonsbury-Martin et al. (1988) that ears exhibiting several SOAEs had no detectable regions of local- t By inference, then, hearing sensitivity in general should be somewhat better in MZ females (especially dark-eyed females) than in other people, and somewhat worse in OSDZ females than other females. To verify these predictions, psychophysical tests more precise than the audiometric screening done here would be required. It is sometimes asserted that hearing is hypersensitive in the vicinity of an SOAE, but care must be taken to understand the basis for this assertion. The existence of a minimum in the microstructure of the audiogram (e.g., Zwicker and Schloth, 1984; Long and Tubis, 1988) near the frequency of an SOAE is not good evidence for a localized hypersensitivity of the cochlear structures. The reason is that the presence of a test tone close to the frequency of an SOAE can lead to a beat-like interaction between the tone and the SOAE, and it is likely that the high detectability of such tones is attributable to the presence of this acoustic interaction, not to a hypersensitivity of the cochlear structures at the location of the SOAE. Better evidence for localized high sensitivity (but not necessarily hypersensitivity) of the cochlear structures is the fact that the spectra of click-evoked and stimulus-frequency OAEs often have local maxima--some at SOAE frequencies and some not (Probst et al., 1991). That is, minima in the microstructure of the audiogram near an SOAE probably have a different basis from maxima in the CEOAE and SFOAE spectra elsewhere, and only the latter should be interpreted as evidence for localized high sensitivity in the cochlear structures.

12 192 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) ized damage in those structures known to be susceptible to damage from exposure to intense sounds. Typically in mammalian species, the cochlear outer hair cells (OHCs) are arrayed in three, extraordinarily regular, rows along the entire length of the basilar membrane. However, in some higher primates, including humans, a fourth or even fifth row of OHCs can exist over short stretches of the basilar membrane (Lonsbury-Martin et al., 1988), and these additional OHCs are most common at the apical (low-frequency) end of the cochlea. Geisler (1986) suggested in passing that the occasional fourth row of OHCs might be the physiological basis for SOAEs. This idea is appealing in part because both the extra OHCs and SOAEs are seen only in higher primates and because both are primarily low-frequency phenomena. Further, OHCs are motile elements that change shape in response to displacement of the cochlear partition and other influences (Brownell et al., 1985), and it is plausible that the normal micromechanics might be upset in a localized region of the cochlea having extra motile elements. Lonsbury-Martin et al. (1988) made careful histological examinations of the cochleas of a rhesus monkey that had detectable SOAEs and compared them with control cochleas. They found considerable irregularity in the placement and orientation of the OHCs in both the emitting and control ears, which made it impossible for them to unequivocally identify a feature of cochlear structure that was perfectly correlated with the existence of SOAEs. However, they did note the presence of greater irregularity in the OHCs and supporting structures, and abrupt interruptions in the fourth row of OHCs, in the ears that emitted SOAEs. Probst et al. (1991) suggested that the slightly smaller physical dimensions of female cochleas (e.g., Sato et al., 1991) may lead to greater irregularity in the patterns of OHCs and related structures and, thereby, to the greater prevalence of SOAEs in females than males. A study of the cochleas of people exhibiting numerous SOAEs (such as MZ females) might be helpful in establishing the physiological basis for SOAEs. Pickles (1982, p. 128) suggested that an excess of intracellular calcium caused by defective calcium channels in an outer hair cell might lead to actin/myosin interactions that would produce displacements of the cell's stereocilia and thence the cochlear partition. A succession of these contractile responses might produce a self-sustaining loop of excitation for that hair cell and eventuate in an SOAE. In a weak test of this idea, a drug that blocks calcium channels (verapamil) was administered to a subject having an SOAE, and the SOAE was not affected (McFadden and Plattsmier, 1984). The mammalian cochlea receives a supply of efferent fibers that originate in the hindbrain and terminate on the hair cells and the primary auditory fibers (Warr, 1992). The contribution to everyday hearing from this efferent system is still unknown, but it is widely regarded to be a source of inhibition in the cochlea. Some of these efferent fibers originate contralaterally and contact OHCs primarily. When these crossed efferents are activated, SOAEs and other types of otoacoustic emissions can be temporarily reduced or eliminated (Mountain, 1980; Mott et al., 1989). Accordingly, were the strength of efferent inhibition low in some localized cochlear region, or in an entire ear, then the resulting 'disinhibition' might lead to more SOAEs being exhibited. That is, SOAEs might result from local irregularities in efferent input, and the sex, ear, and pigmentation differences seen in SOAE expression might be attributable, in part, to (genetically determined?) differences in the general strength of the efferent flow into different cochleas (McFadden, 1993a). Finally, it is possible that small differences in the temperature or the blood supply in individual cochleas are responsible, in part, for the ear, sex, and individual differences in SOAE expression (e.g., Wilson, 1986) Timing of prenatal ecents possibly related to SOAEs While it is unknown exactly what cochlear features are affected by prenatal exposure to such hypothesized SOAE-altering factors as high levels of androgens, perhaps inferences can be drawn about the timing of these effects. MZ twins become separate embryos soon after conception. By one estimate, about one-third of all MZ twins have divided into separate embryos within the first 5 days after conception, about two-thirds divide between days 5 and 10, and only about 4% divide later than days post-conception (Bulmer, 1970, p. 33). One of the structures likely to be involved in the prenatal determination of SOAE expression is the neural crest, for it is from this set of cells that the auditory system and all pigmented cells develop. The neural crest begins to emerge about 3-4 weeks after conception (Kandel and Schwartz, 1985, p. 245, p. 730) or at least 1-2 weeks after all MZ twins have separated into two embryos. Finally, male fetal testosterone levels reach maximum during weeks post-conception, after which they decline to values indistinguishable from those seen in female fetuses (Reyes et al., 1974). These facts suggest that potential SOAEs are eliminated in males (including MZ males), and in females having male co-twins, during weeks For comparison, by week 10 post-conception, the embryonic human cochlea has achieved its full 2.5 turns and scala tympani and scala vestibuli have been formed, although adult dimensions are not achieved until the fifth month of prenatal development and functional maturity not until the seventh or eighth gestational

13 D. McFadden, J.C. Loehlin / Hearing Research 85 (1995) month (Lavigne-Rebillard and Pujol, 1990; see reviews by Hecox, 1975, and Walsh and McGee, 1986). The inner and outer hair cells are identifiable by week 12 post-conception, and they are preceded by the emergence of identifiable auditory nerve fibers. In general, the appearance and maturation of inner hair cells precedes that of outer hair cells, the development of synapses precedes that of stereocilia, the cochlear base (high frequencies) matures before the cochlear apex (low frequencies), and the cochlear efferent system is far slower to mature than the afferent system (Lavigne-Rebillard and Pujol, 1990). At maturity, male cochleas are about 13% longer than female cochleas (Sato et al., 1991). Both male and female embryos are exposed prenatally to rather high levels of androgens (that are secreted by the mother as well as by male embryos), and large individual differences--within and across the sexes--are known to exist in these prenatal androgen levels (Reyes et al., 1974; Weisz and Ward, 1980). Accordingly, it may be that normal variation in the androgen levels to which both male and female embryos are exposed contributes to the wide variation observed in SOAE expression. That is, for males and females, and twins and non-twins, perhaps the likelihood of our observing SOAEs is diminished by the existence of especially high androgen levels during specific periods of prenatal development--either by chance or as a result of such factors as maternal stress (vom Saal et al., 1990). Hines (1982) and Kimura (1992) have discussed the prospect that prenatal hormonal exposure may be responsible for certain sex differences in behavior. As noted previously (McFadden, 1993b), confirmation of the idea that exposure to high levels of androgens is responsible for the sex difference in expression of SOAEs may be possible by studies of SOAEs in people who experienced atypical androgen exposure prenatally--e.g., cases of testicular feminization (Cummings, 1991, p. 108), congenital adrenal hyperplasia (Berenbaum and Snyder, 1995), and other maladies. While additional work will be necessary to establish the reality of the higher prevalence and number of SOAEs in MZ females apparent here and in the data of Russell (1992), there may be some value in a brief, provisional discussion of possible mechanisms underlying such an outcome. One logical possibility is that MZ females are prone to a mechanism that adds SOAEs in some way. We find this possibility difficult to accept, however, because the process of MZ twinning occurs so early in development relative to the emergence of the precursors to the cochlea proper. Alternatively, one might argue that the high prevalence of SOAEs in MZ females is simply attributable to their having been co-gestated with another female--and thereby possibly exposed to higher levels of female hormones prenatally --but that just changes the problem into explaining the lower prevalence of SOAEs in SSDZ females than in MZ females. We find it more parsimonious to presume that various factors act to eliminate potential SOAEs and that, for some reason, one or more of these prenatal moderating influences fails to act effectively in MZ females, thereby 'leaving' more SOAEs to be expressed than in other people Possible genetic mechanisms In Table 3, the SSDZ twin correlations are less than half the MZ twin correlations. Such a correlational pattern is quite common with human behavioral traits --for example, in the area of personality (Loehlin, 1992b)- and it suggests some degree of non-additive action in the genes affecting the trait in question. Genetic non-additivity, if present, could represent interactive effects of genes at a single locus on the chromosome ('dominance') or across loci ('epistasis'). If the genetic variance affecting a trait is largely nonadditive, this may be evidence that the trait has been under recent natural selection, as selection tends first to decrease additive genetic variance (Mather, 1966). However, as noted, the sample sizes in the present study were small, and the data did not depart significantly from an additive model. Moreover, the data of Russell (1992) appear quite consistent with an additive model. Therefore, any detailed speculation concerning implications for the evolutionary history of SOAEs appears premature. The differing prevalence of SOAEs in males and females led Bilger et al. (1990) to speculate that SOAEs might represent an X-linked trait. As they noted, pedigree studies would provide the most direct test of such an hypothesis, which makes strong predictions about father-son transmission (sons get their X-chromosomes only from their mothers, never their fathers). Our own proposal involves 'sex-influence': the differential expression of SOAEs in the two sexes is postulated to be due, in part, to the effect of male hormones during prenatal development. We also suspect that a number of genes will ultimately prove to be involved in the expression of SOAEs, meaning that, for the present, studies directed to summarizing their effects--such as twin and family studies--will be more fruitful than direct pedigree searches for individual genes. But that, of course, is for future research to determine. One question we have considered is whether the specific locations along the cochlear partition (and hence the acoustic frequencies) of individual SOAEs might be genetically determined. If so, the acoustic frequencies of the SOAEs in the ears of MZ co-twins, or in the two ears of an individual, should resemble each other more than those in the ears of DZ co-twins, for example. A priori, this possibility appears rather

14 194 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) unlikely because a large number of genes devoted to the individual inheritance of specific SOAEs would seem to be an evolutionary extravagance, especially for a trait without obvious selective advantages. Nevertheless, we used two strategies to explore this question. One involved the development of ways to characterize the overall resemblance of the pattern of frequencies in two ears, a task made difficult by the fact that any two ears are likely to have different numbers of SOAEs, and thus the matching process can be problematic. Two indices that we tried for this purpose, the Average Minimum Frequency Ratio (AMFR) and a coefficient due to Gini, are described in an Appendix. The second strategy involved tabulating very close matches (frequency ratios of less than between pairs of SOAEs from relevant pairings of ears) and then comparing the number of these with the number obtained when the same number of SOAEs were randomly assigned within the range of acoustic frequencies studied (for, of course, the greater the number of SOAEs in a pair of ears, the greater is the likelihood of obtaining close matches by chance). Neither of these approaches provided convincing evidence for a genetic contribution to individual SOAE frequencies. So, the most parsimonious working hypothesis appears to be that the genes involved act on general properties of the relevant anatomical structures, such as their relative sizes, the development of extra outer hair ceils, etc.- properties that affect the likelihood of SOAEs occurring at all--and that the locations of the particular anomalies that lead to individual SOAEs are chance consequences of local conditions during the developmental process. Comment There are recent discoveries in the study of the co-evolution of parasites and hosts that have implications for all heritability research, and while they probably do not affect the current findings, they are sufficiently interesting to warrant mention. Recent evidence indicates that certain diseases and maladies that previously have been shown to be heritable (e.g., multiple sclerosis and juvenile diabetes) involve an autoimmune response (Steinman, 1993). Everyone inherits an immune system that produces one of a set of possible responses to a particular pathogen, but some of those characteristic responses are lamentably inappropriate because they give rise to an autoimmune reaction that then leads to the disease or malady. The apparent heritability is partly genuine (inherited similarities in the immune systems of the people being studied) and partly spurious (similarities in their individual histories of exposure to specific pathogens)--but in neither case is it due to genes controlling the trait directly. Were a mechanism of this sort operating to produce (or eliminate) SOAEs, the exposure to the pathogen would have to occur prenatally (because SOAEs are present at birth with the same ear and sex differences seen in adults, and they appear to be highly constant through life), and it would have to have a lower probability of affecting males (and their co-twins) than females, and a higher probability of affecting both members of MZ than DZ pairs. It seems highly unlikely to us that the expression of SOAEs will ultimately prove to be based on an autoimmune reaction, although the array of maladies known to involve aspects of autoimmunity has increased dramatically in recent years (Steinman, 1993), and the auditory system may not remain exempt Relationship to eye color The initial motivation for partitioning the present data by eye color was the first author's abiding interest in the possible relationship between peripheral pigmentation and auditory function. The possibility of a link between peripheral pigmentation and SOAE prevalence was not unreasonable given both the common association between pigment anomalies and congenital deafness, and the fact that the neural crest gives rise embryologically both to pigmented cells and the auditory system (e.g., Brown, 1973; Cable et al., 1992). Eye color has been related to melanin concentration in the inner ear (Bonaccorsi, 1965), various drugs that affect hearing bind to melanocytes (Lyttkens et al., 1979), and some research suggests that dark-eyed people are more resistant to noise-induced hearing loss than are light-eyed people (for a review, see McFadden and Wightman, 1983). After this experiment had begun, Whitehead et al. (1993) reported that the mean number of SOAEs per ear was 3.3, 2.1, and 1.2 in Negroes, Asians, and Caucasians (with light eyes) respectively, in support of the possibility that some aspect of peripheral pigmentation is related to SOAE expression. Also, Russell (1992) reported a higher prevalence of SOAEs in Afro-Americans than in Euro-Americans. While there was a trend in our data toward more SOAEs in dark-eyed than light-eyed subjects, it did not achieve statistical significance. Perhaps eye color is not as closely related to SOAEs as is skin color, or perhaps some racial differences other than pigmentation are also important. It may be relevant that our sample contained a number of Hispanic subjects, who are predominantly dark-eyed but typically lack the degree of skin pigmentation of African-Americans. Whatever the explanation, a breakdown of the present data by race is not feasible because that information was not collected Other factors and controls Several factors known to affect SOAEs were not well-controlled in this study, and the likely conse-

15 D. McFadden, J.C. Loehlin /Hearing Research 85 (1995) quence is an underestimate of the number of SOAEs, or of the similarity of SOAEs in particular pairs of ears. First, a number of our subjects admitted having used one or more over-the-counter drugs for allergies or other minor maladies in the 24 hours preceding our measurements. Most drugs have yet to be tested for their effects on SOAEs; however, in all cases to date, the drugs that have affected SOAEs have reduced or eliminated them (e.g., McFadden and Plattsmier, 1984; Long and Tubis, 1988; McFadden and Pasanen, 1994; but compare Stypulkowski and Oriaku, 1991). Second, a few ears having multiple emissions are known to exhibit specific subsets of SOAE frequencies at some times and different subsets at other times (Burns et al., 1984). These ears move unpredictably from one quasistable state to the other. If identical twins both possessed the same two quasi-stable states, but were weby chance--to sample their emissions when the ear of one twin was in one state and the ear of the other in the second state, the correspondence between number of SOAEs (and their acoustic frequencies) for that pair of co-twins would have appeared worse than had both twins been in the same quasi-stable state. Third, to the extent that some of the detected SOAEs were distortion products, resulting from the non-linear interaction of two, relatively strong SOAE 'primaries' (Probst et al., 1991), then the absence, or a small difference in the frequency, of one of the 'primaries' in one co-twin might also have eliminated the distortion-product SOAE--thereby further decreasing similarity. Finally, since the effects of menstruation on SOAEs are small (e.g., Bell, 1992), no control was attempted over this variable. As an added check on possible artifacts, we obtained correlations for number of SOAEs across pairs of non-twins tested simultaneously. Correlations for corresponding ears of 13 female/female and 11 male/male pairs were calculated. None was significantly different from zero by a permutation test; in fact, only one of the four was even positive. 5. Summary and conclusion The data and analyses presented here suggest that about 75% of the individual variation in the expression of spontaneous otoacoustic emissions can be attributed to genes, with the genes involved probably acting additively. By comparison, previous twins research studying well-measured physical traits has produced some estimates of heritability that are higher, and some that are lower, than ours: height, 0.90; fingerprint ridge count, 0.94; weight, Among behavioral traits, IQ is near 0.80 (in adults), but more specialized cognitive abilities and personality traits tend to lie in the 0.30 to 0.50 range (Plomin et al., 1990). The large differences in SOAE expression seen between the sexes, and across individuals, are likely attributable--at least in part--to differences in prenatal development, especially to prenatal differences in exposure to androgens. These prenatal influences are conceptualized as acting primarily to eliminate potential SOAEs, not to produce additional ones. We do not believe there was evolutionary pressure to produce SOAEs per se, but, rather, that SOAEs are an epiphenomenon covarying with other auditory characteristics, such as hearing sensitivity, which were subject to evolutionary pressure. Of considerable interest is why some individuals and groups are so prone to whatever processes underlie SOAEs, and how that tendency is realized. Acknowledgements All of the data were collected by G.L. Dykstra, R. Mishra, E.G. Pasanen, and J.G. Sirianni, while S.D. Casto and N.L. Callaway provided invaluable help with scheduling and recruiting subjects. E.G. Pasanen helped with instrumentation and programming. This work profited from conversations with, and assistance from, R.C. Bilger, F.S. vom Saal, W.S. Geisler, N.G. Waller, and J.M. Horn, as well as from the comments of three anonymous reviewers. R.R. Cocke and D.D. Thiessen brought to our attention the additional complication that autoimmune reactions create for the interpretation of heritability estimates. We thank W.S. Geisler and L.A. Stern for making the spectral measurements on our artificial eyes. This research was supported by grant DC from the National Institute on Deafness and Other Communication Disorders (NIDCD) awarded to DM. Appendix 1 On similarities of SOAE frequencies As noted in the Discussion section, attempts were made to measure the similarity of the acoustic frequencies of the SOAEs in different combinations of ears-the question being whether there was evidence for heritability of specific SOAE frequencies, not just for the number of SOAEs exhibited. Two statistical procedures were used. The first was a version of Gini's quadratic index of dissimilarity (Pietra, 1925). This statistic expresses the difference between two ordered series of different lengths by extending them both to a common length, and then taking the root-mean-square difference between corresponding terms in the extended series. Extended series of this sort can always be achieved by

16 196 D. McFadden, ZC. Loehlin /Hearing Reseamh 85 (1995) listing each term in the first series n 2 times and each term in the second series n 1 times, where n I and n 2 are the respective lengths of the two initial series. Note that only pairings in which both ears have at least one SOAE can contribute to the coefficient resulting from this method. To understand the second procedure for comparing the similarity of the acoustic frequencies of SOAEs in paired sets of ears, first consider the following procedure: For each SOAE frequency in one ear (the test ear), find the SOAE frequency in a second (comparison) ear that is algebraically closest, and calculate the absolute difference in Hertz. For convenience, call this the minimum difference for that particular SOAE. Once minimum differences of this sort have been obtained for every SOAE in that test ear, calculate the mean of those minimum differences and use that value to typify the similarity of the test ear with the comparison ear. For convenience, call this the average minimum frequency difference (AMFD) across all SOAEs in a particular ear. [Note that similar calculations with the test and comparison ears reversed are necessary to determine the similarity in the opposite direction because only when both ears of a pair each have only one SOAE is the (average) minimum difference necessarily the same for the initial and reversed cases.] A problem with the AMFD calculation described is that frequency location along the basilar membrane varies logarithmically with linear distance (e.g., Greenwood, 1990), meaning that algebraic differences in SOAE frequency will exaggerate the difference in relative location from which a pair of SOAEs originate. If one assumes that similar heredity would tend to produce similar effects at particular locations along the cochlear partition, then a calculation employing log frequencies, or, equivalently, the ratios of frequencies would be preferable to the AMFD calculation described. Accordingly, calculations like the AMFD described above were performed using frequency ratios, and an average minimum frequency ratio (AMFR) was determined for each test ear. (Specifically, for all calculated frequency ratios less than 1.0, the reciprocal was obtained, and then the frequency ratio closest to 1.0 taken as the minimum for that comparison of SOAEs. A simple average of these minimum frequency ratios was taken as the AMFR for a test ear, and these averages were themselves averaged across subjects.) The Gini and AMFR procedures agreed fairly well. Typical correlations between them for our data lay in the range of 0.80 to While the calculation of Gini dissimilarity indices and AMFRs has considerable potential for assessing the similarities in the acoustic frequencies of the SOAEs in specific pairs of ears, in practice their full potential could not be realized because of the relative dearth of SOAEs in SSDZ twins, males, and left ears, as compared to MZ twins, females, and right ears. Whenever a comparison ear has no SOAEs, no AMFR can be determined for the test ear. Thus, the effective size of any sample of twins or non-twins is diminished, and this problem is especially acute for those comparisons involving ears having relatively few SOAEs. Further, the more SOAEs a test and comparison ear have, the greater is the potential for a small AMFR value to result for that comparison by chance--emphatically a problem when comparing MZ females with other subject groups. 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